Ingestible composition having hydrogen peroxide with an increased half-life

ABSTRACT

The present invention is directed to a method and a composition for producing and using a plant-based biocidal solution. The plant-based biocidal solution contains a bioactive material and a plant-based substance formed from the cellular material of a plant. The plant-based substance is capable of binding to the bioactive material. In some embodiments, the bioactive material is hydrogen peroxide. The hydrogen peroxide can be added exogenously or generated endogenously. In accordance with further embodiments, the plant-based biocidal solution can be applied to a target, thereby impairing the target. In some embodiments, the target can be a pathogen. In accordance with another embodiment, the plant-based substance of the plant-based biocidal solution can form a microscopic cluster, a complex, or an aggregate for providing sufficient bioactive material to overcome the defense mechanism of the target.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/463,992, filed Mar. 20, 2017, which is a continuation of U.S.application Ser. No. 14/221,500, filed Mar. 21, 2014, now U.S. Pat. No.9,636,361, which is a continuation of U.S. application Ser. No.12/868,634, filed Aug. 25, 2010, now U.S. Pat. No. 8,722,116, which is acontinuation of U.S. application Ser. No. 12/317,638, filed Dec. 23,2008, now U.S. Pat. No. 8,734,867, which claims the benefit of U.S.Provisional Application No. 61/009,484, filed Dec. 28, 2007, eachapplication of which is hereby incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates generally to materials and systems that provide apreservative and/or a medicinal effect. More specifically, thisinvention relates to anti-bacterial, anti-infective, anti-microbial,anti-fungal and antibiotic materials and systems that utilize materialobtained from plants.

BACKGROUND OF THE INVENTION

Consumer products and environmental mandates have created a large demandfor natural biocides. There are a number of commercial extracts made ofplants, notably essential oils, that provide antimicrobial activity.However, all current natural antimicrobials are limited by somecombination of low potency, high cost, toxicity, taste, odor and colorof the extracted compounds at their minimum effective concentrations.Synergistic combinations of essential oils have met with some success,but their performance still pales compared to common synthetic biocides.The pharmakinetics of all current commercial antimicrobials is stillbased on dilution of individual molecules.

Plants have various mechanisms for delivering localized and concentratedimmune responses against pathogens. Plants cannot rely on generaldiffusion of antimicrobial compounds throughout their tissues. Effectiveprotective concentrations would be systemically toxic. When plants areexposed to external stress, an almost universal defense mechanism is thelocal expression of reactive oxygen species (ROS) that initiatecompounds rapid formation of physical barriers as well as mountingdirect attack against the invading pathogen. These ROS include hydrogenperoxide (H₂O₂), superoxide (O₂ ⁻), singlet oxygen (¹O₂*), and hydroxylradical (.OH).

These radicals damage the cell walls of pathogens on contact or create ahyperoxygenated environment that the cell cannot tolerate. They also caninitiate reactions of alkaloids, terpenes, phenolics, peptides or otherastringent compounds, to aggressively bind and immobilize amino acids ofthe plant and pathogens.

The cell walls of bacteria and fungi are protected by peroxidase,catalase, and other enzymes that scavenge ROS. Therefore, an effectiveoxidative attack on a pathogen requires providing a sufficientconcentration of ROS molecules to overwhelm the pathogen's defenses.

The cell walls and membranes of eukaryotic organisms are populated withperoxisomes. Subcellular organelles, which are rich in enzymaticproteins, carry out a wide range of functions including β-oxidation offatty acids, glyoxylate metabolism, and metabolism of reactive oxygenspecies. H₂O₂ producing enzymes NAD(P)H oxidase, oxalate oxidase, andglucose oxidase, are found on the peroxisomal membrane. Peroxisomescontain antioxidant molecules, such as ascorbate and glutathione, thecell's principal H₂O₂ degrading enzyme-catalase, and a battery ofantioxidant enzymes, including superoxide dismutase, ascorbateperoxidase, dihydro- and monohydroascorbate reductase, glutathionereductase. These tightly regulate the amount of H₂O₂ accumulation inhealthy plant tissue. Changes in activities of these enzymes arecorrelated with many situations in which plants experience stress.Accordingly, peroxisomes have been suggested to play important roles indefense against abiotic and biotic stress in plants. Mitochondria andchloroplasts also use H₂O₂ as a transduction medium. Superoxides arealso converted in the organelle matrix.

Reactive oxygen species (ROS) can destroy invading microorganisms bydenaturing proteins, damaging nucleic acids and causing lipidperoxidation, which breaks down lipids in cell membranes. Both plantcells and pathogens are protected, at least in part, from ROS byenzymatic and non-enzymatic defense mechanisms.

Defense against its endogenous ROS as well as a pathogen ROS attack isbelieved to be provided by the scavenging properties of antioxidantmolecules found in the organelles and the cell membranes. Superoxidedismutases (SODs) catalyze the reduction of superoxide to hydrogenperoxide. Hydrogen peroxide is then decomposed to H₂O by the action ofcatalases and peroxidases. A certain concentration of H₂O₂ also diffusesinto the intracellular matrix and is released by lysis or mechanicalrupture of cells. Cell disruption causes H₂O₂ to come in contact withseparately compartmentalized polymers and initiates rapid cross-linkingof cellular proteins to form a protective barrier at localized stresssites. The in-vivo anti-bacterial efficacy of antibiotics encapsulatedin synthetic liposomes was demonstrated to be four times more effectivethan the free systemic application (Halwani and Cordeiro, et at, 2001).There is much ongoing research on imparting improved transgenic H₂O₂defenses to commercial crops, genetically modified organisms to producenew antimicrobial compounds, and new botanical sources of antimicrobialextracts. Animal macrophages are another example of specialized immunemechanisms for ROS attack on pathogens. There is a clear advantage tolocalized defensive response over systemic diffusion of antimicrobialchemistry.

Hydrogen peroxide is a common and effective broad spectrum disinfectant,which is notable for its ideal environmental profile (H₂O₂ decomposesinto water and oxygen) and low toxicity. It is an ubiquitousmultifunctional factor in both plant and animal immune and metabolicprocesses. Hydrogen peroxide is generally regarded as safe (GRAS) by theUSDA for use in processing foods when the concentration is less than1.1%. H₂O₂ that has a concentration of 3% is commonly used for topicaland oral disinfectant. Commercially produced H₂O₂ is syntheticallyproduced but identical to that produced in cells and has been acceptedworld-wide for processing nearly every industry. It is an excellentbroad spectrum antimicrobial, but it is too indiscriminating andvolatile for effective use as a product preservative.

The ability to withstand oxidative attack is generally a function of theorganism size. Most pathogens are small and more susceptible to ROSdamage than plant and animal cells. Once the pathogen is depleted of ROSdegrading molecules, further oxidation can damage the cell membrane,causing cell death. This is a completely different mechanism than theblocking of metabolic transduction sites and other highly specificmolecular interactions of antibiotics that are becoming alarmingly lesseffective as bacteria adapt and become resistant.

The tissue of many succulents has a long history of use in traditionalmedicine as antimicrobial wound dressings and for other medicinalpurposes. Aloes are widely cultivated and processed for a variety ofpurposes. Several species of cacti are less widely commercialized butequally valued in traditional medicine and as a food source. Plantsevolved in harsher environments, such as the dessert succulents, tend tohave enhanced capacity to produce hydrogen peroxide in response tobiotic and abiotic stresses. Cleanly sliced fresh pieces of cacti andaloe plants are traditionally effective against infection largely due tothe H₂O₂ expression in the plant tissues in response to its injury.Commercially processed aloe gels generally lose their antimicrobialactivity.

U.S. Patent Application No. 2002/0034553 teaches a composition of Aloevera gel, Irish moss and approximately 3% hydrogen peroxide where thealoe vera primarily forms a gel holding the ingredients together in anointment or lotion which may be applied directly to a cleansed infectedor irritated skin tissue area. The application relies on a conventionalbulk concentration (1.5%) of H₂O₂ to provide an oxygen-rich environment,and it makes no specific teaching regarding functional interactions ofH₂O₂ and the enzymatic or other cellular chemistries of the plantfractions.

U.S. Pat. No. 6,436,342 teaches an antimicrobial surface sanitizingcomposition of hydrogen peroxide, plant derived essential oil, andthickener. However, it does not teach interaction between components.

U.S. Pat. Nos. 5,389,369 and 5,756,090 teach haloperoxidase-basedsystems for killing microorganisms by contacting the microorganisms, inthe presence of a peroxide and chloride or bromide, with ahaloperoxidase and an antimicrobial activity enhancing α-amino acid.Although highly effective antimicrobials, the systems cannot generallybe considered natural products and the components must be separatelystored or packaged in anaerobic containers to preventhaloperoxidase/peroxide interaction and depletion prior to dispensingfor use.

U.S. Pat. No. 5,389,369 teaches an improved haloperoxidase-based systemfor killing bacteria, yeast or sporular microorganisms by contacting themicroorganisms, in the presence of a peroxide and chloride or bromide,with a haloperoxidase and an antimicrobial activity enhancing α-aminoacid. Although the compositions and methods of U.S. Pat. No. 5,389,369have been found to be highly effective antimicrobials, the componentsmust be separately stored and maintained in order to preventhaloperoxidase/peroxide interaction and depletion prior to dispensingfor use.

The above references describe the application of various oxidativeantimicrobials in a free liquid dispersion. Ability of a solution offree soluble biocidal compounds to effectively kill pathogens isdetermined by the probability of individual molecular interactions withthe pathogen. This ability rapidly diminishes with volumetric dilutionand consumption of the active solute.

For this reason, application of diffuse free active chemicals insolution is grossly inefficient at killing bacteria and fungi, yet thisis predominantly how antimicrobials are formulated into commercialproducts for topical therapeutics, personal care products, commercialand industrial sanitizers, and sanitation or preservation of food andwater. This method demands extraction processes that highly concentrateactive chemicals. To obtain adequate microbial suppression, productformulations commonly require higher concentrations of these chemicalsthat would be toxic to the tissues of plants of origin. There are someexamples of encapsulation of essential oils for stabilization offragrance, and commercially available synthetic liposomes for targetedintravenous drug delivery, but there are no commercial examples ofex-vivo generation of unencapsulated plant material complexes forimproved antimicrobial efficiency.

FIG. 1 illustrates a traditional biocidal solution 100. The workingprinciple of traditional biocidal solution is based on free liquiddispersion. The effectiveness of the biocidal compounds solution isdetermined by the probability of individual molecule that encounterswith the pathogen. The target 102 is within the solution 100 whichcontains free liquid dispersed hydrogen peroxide 104.

Therefore, there exists a need for compositions and methods of preparingmicroscale antimicrobial complexes or aggregates of stable activechemistries that provide an efficient means of concentrating the assaulton pathogenic organisms. Ideally, such antimicrobial complexes should befast acting with minimal host toxicity and with maximal germicidalaction. The compositions should be naturally derived, easy to deliver orformulate, and should not cause damage to host tissue or common surfaceson contact. Depending upon the strength of composition and the timeinterval of exposure, the compositions should produce antisepsis,disinfection, or sterilization at lower molar concentrations thantypical free active chemicals in solution. Such compositions will haveutility as an efficient means of controlling microbial population foranti-infection, sterilization, deodorization, sanitation, environmentalremediation, preservation of topical products, and safety andpreservation of food and water.

SUMMARY OF THE INVENTION

The present invention describes compositions, applications, applicationmethods and methods of producing a biocidal substance with a substrateof biologically reactive material. In some embodiments, the biocidalsubstance includes plant-tissue aggregates, extracted polymers, orcombinations thereof. In some embodiments, the biocidal substance with asubstrate of biologically reactive material creates high localizeddensity of bioactive sites for improving microbicidal efficiency andastringent effect.

In one embodiment of the invention, the method of forming and thecomposition of a plant-based biocidal solution includes a bioactivematerial and a plant-based substance formed from the cellular materialof a plant capable of binding to the bioactive material. In someembodiments, the interaction between the plant-based substance and thebioactive material stabilizes the bioactive material. The combination ofthe plant-based substance and the bioactive material provides a stablesource of providing bioactive material. In some embodiments, thebioactive material is a substrate of compounds of reactive oxygenspecies. Alternatively, in some embodiments, the bioactive material ishydrogen peroxide. The hydrogen peroxide can be generated endogenouslyor exogenously. The exogenously added hydrogen peroxide can be obtaineddirectly from commercially available sources. In some embodiments, suchhydrogen peroxide has a concentration of 1%-90% hydrogen peroxide inwater. Alternatively, the hydrogen peroxide has a concentration of25%-50% hydrogen peroxide in water. The endogenous generation ofhydrogen peroxide can be achieved by measured gross cutting or otherphysical abiotic stressing of a metabolically viable harvested plantstructure or controlled wounding of a pre-harvest plant to activate anexpression of an increased H₂O₂ acting compound. Alternatively, in someembodiments, the bioactive material is generated by the degradation ofadded ozone (O₃) by an active dismutase in the complex or solution, orin combination with direct addition of the H₂O₂.

In some embodiments, the plant-based substance, cellular materials, andplants are obtained naturally or artificially. In some embodiments, theplant-based substance is formed from a cellular fragment, a multivalentpolymer, an oligomer, an intact cell, a lignin, a subcellular organelle,a membrane fragment, a soluble protein, a polysaccharide, a phenoliccompound, a terpene, an enzyme, and a denatured proteinaceous fragment.In some embodiments, the cellular material comes from a cell of a plantwith a hydrogen peroxide acting enzyme on the cell membrane, a membranebound organelle, or a tissue with the ability to fix and significantlyincrease the half-life of hydrogen peroxide or other oxygen radicalwhile preserving its bio-reactivity. Most higher plants have some degreeof ROS generation and preservation capability in their tissues. In someembodiments, the plant is a species from of the family of Cactaceae,Agavaceae, or Poacea. In some embodiments, the plant is a species with ahistory of food or medicinal application or Generally Regarded As Safe(GRAS) by the U.S. Department of Agriculture (USDA) or U.S. Food andDrug Administration (FDA). In some embodiments, the plant-based biocidalsolution can include water, gas, supercritical fluid, organic solvent,inorganic solvent, or any combination thereof.

In accordance with further embodiments, the bioactive material-degradingenzyme, such as catalase and peroxidase, contained in the cellularmaterial needed to be processed to be at least partially inactivated.This can be accomplished by desiccating, blanching, heating of driedmaterials, exposing to UV radiation, freeze-thaw cycling, heating orboiling in a solution of water, storing processed or partially processedfor natural degradation with time, or exogenously adding of a chemicalenzymatic inhibitor.

In some embodiments, the plant-based biocidal material is a naturalproduct. The plant-based biocidal material is able to be used alone, incombination with other oxidizers, material, or in synergisticinteraction with additional exogenous or endogenous plant-derived orsynthetic antimicrobials. In some further embodiments, the plant-basedbiocidal material has an effective concentration sufficiently low todilute a non-functional or undesirable component or characteristic insolution to a sub-functional or sub-concern level.

Another embodiment of the invention is the use of the plant-basedbiocidal material to impair a target. The use of the plant-basedbiocidal material is achieved by taking the plant-based substance formedfrom a cellular material of a plant with a bioactive material andapplying the plant-based substance with the bioactive material to atarget, such as a pathogen. The applying of the plant-based substancewith the bioactive material is able to deliver high localizedconcentration of the bioactive material to the target. Such applicationcan be for a purpose of providing direct biocidal activity or forbiocidal preservation of the formulation. Furthermore, such applicationcan also be applied to a beneficial effect to human or animal woundhealing, such as exudate control, wound closure, and rapid scabformation attributable to the aggressive bio-oxidative and protein crosslinking capacities of H₂O₂ by itself, or in combination withpotentiating endogenous and or exogenous co-factors.

Another embodiment of the invention is the method of using theplant-based biocidal material. The use is achieved by taking aplant-based substance formed from a cellular material of a plant with abioactive material, forming a microscopic cluster, a complex, or anaggregate from a suspension of the plant-based substance, and applyingthe microscopic cluster, the complex, or the aggregate to a target, suchas a pathogen, thereby impairing the target. In some embodiments, theimpairment of a target can be oxidative damage. In other embodiments,applying the microscopic cluster, the complex, or the aggregate toimpair a target is for the purpose of stable binding of a denselocalized concentration of hydrogen peroxide to enact a nearlysimultaneous oxidative attack with sufficient number and rate ofreactions to overwhelm oxygen scavenging and enzymatic Reactive OxygenSpecies (ROS) defenses of pathogens or a combination of isolation,immobilization and ROS attack.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a solution containing free liquid dispersion ofhydrogen peroxide taught by prior art.

FIG. 2 illustrates a plant-based biocidal material in accordance withthe present invention.

FIG. 3 illustrates a flowchart of a method of combining plant-basedsubstance and a solution containing bioactive material in accordancewith the present invention.

FIGS. 4A and 4B illustrate uses of plant-based biocidal solution inaccordance with the present invention.

FIG. 5 illustrates another use of plant-based biocidal solution in theform of microscopic clusters, complexes, and aggregates in accordancewith the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The antimicrobial potency of a liquid composition is a function of thelocal density of H₂O₂ or other bio-active molecules within reactiveproximity of the pathogen. The biocidal effectiveness of each individualaggregate does not substantially change with gross dilution though theprobability of aggregate-pathogen encounter, and therefore the kill ratein time will be an inverse

function of gross dilution. In the present invention, the antimicrobialaggregate or plant-based substance has the potential to deliver a highlocalized concentration that is more efficient at killing organisms thana diffuse dispersion of the same molar concentration of free activemolecules. This composition, and method of producing efficientplant-based antimicrobial compositions with extremely low minimumeffective concentration is both novel and valuable in a multitude ofcommercial uses. It will be readily apparent to one skilled in the artthat other various modifications can be made, and the presentapplication is not limited to the use of H₂O₂ or antimicrobial activity.

One embodiment of the composition in the present invention utilizesaspects of oxidative defenses mechanisms that occur in most plants.Plants generate H₂O₂ as a multifunction molecule for both defense andmetabolic functions. Plant cellular structures, such as peroxosomes,mitochondria, and cell membranes, are sites of H₂O₂ generating andregulating enzymes. H₂O₂ related enzymes, such as catalases,peroxidases, oxalate oxidases, glucose oxidases and dismutases, have theability to capture and catalyse reactions with H₂O₂. Even if inactive intheir original function, the structure of these molecules can providelow energy binding sites for fixing H₂O₂ while preserving itsbio-reactivity. Therefore, the present invention uses a plant-basedsubstance formed from the cellular material of a plant to bind variousbioactive material, including H₂O₂.

Certain active enzymes, such as catalases and peroxidases, decomposeH₂O₂ and are undesirable in the compositions of the invention. Theseenzymes are susceptible to degradation; therefore, heat, cold,dessication or time are essential steps used in the process to reducethe population of H₂O₂ reducing enzymes prior to the addition ofexogenous H₂O₂.

The plant-based biocidal solution of the invention can be applied to,but not limited to: cosmetic preservative, food preservative, watersanitizer, persistent surface sanitizer, water preservative,environmental remediation, sewage treatment, medical therapy of wounds,medical treatment of gastric infections, medical treatment of chroniculcers, medical treatment of gingivitis, treatment of halitosis,sterilization of medical instruments, sterilization of contact lenses,sterilization of surfaces, shelf life extension of fresh foods,reduction of bacteria in aquatic farms or aquaria, prophylacticprevention of gastric infections, first aid antiseptics, and treatmentof fungal infections.

Some embodiments of the present invention provide a naturalantimicrobial extract that has fewer adverse toxicological andenvironmental impacts than traditional biocides such as chlorine,phenolics, aldehydes and quaternary ammonium salts.

Alternatively, some embodiments of the invention use only natural planttissues in combination with only exogenous oxidizers approved by theUSDA for food use and GRAS from the following: hydrogen peroxide, ozone.The H₂O₂ containing solution is then diluted by at least 100:1 beforecommercial application. At such dilutions, remaining free H₂O₂ or O₃will preferably spontaneously decompose in a short period of time atroom temperature, leaving only chemical species consistent with thosenative in the plant source.

Some embodiments of the invention obtain sufficient antimicrobialpotency at very high dilutions to reduce the direct or accumulatedeffects of undesirable substances that have toxicity or produce taste,odor, or color. This uniquely allows the use of simplified whole planttissue utilization with reduced or eliminated need for selectiveextraction or isolation processes. This further improves the ability andopportunities for producing compositions that are natural andpotentially organic product or ingredient.

Some embodiments of the invention significantly increase the half lifeof the bound H₂O₂ in its composition as compared to free H₂O₂. Theantimicrobial activity of the 100 ng/ml solution was challenged at 2, 7,17, and 30 days, and exhibited no significant reduction in E. Colikilling capability. After 90 days at room temperature and ambientflourescent light exposure, the original master batch of Pachycereuspecten-aboriginum composition showed no reduction in potency.

Some embodiments of the invention also provide a composition withnon-biocidal benefits in animal and human wound healing. Compositionsprepared from the two species of Cactaceae induce rapid closure of cuts,exudate control, and rapid fibrin scab formation on open abrasions.

Some embodiments of the invention provide a biocidal composition with amechanism of action less prone to resistance selection. Packet oxidativeattack that overwhelms pathogen defenses minimizes weakened survivors topropagate resistance. Some embodiments of the invention provide acomposition with a significantly lower effective concentration than mostsynthetic disinfectants and antibiotics. The tested compositions containless than 10 ng whole dry plant mass/gram of water, and they are 100%effective against a broad spectrum of yeast, gram-positive bacteria, andgram-negative bacteria in vitro. Further, some embodiments of theinvention provide increased safety and dosage margins in animal/humanmedicinal and food applications. Hydrogen peroxide oxidative attack onpathogens is selective against non-eukaryotes and has no accumulativeby-products.

The invention provides a composition for topical application on humanand animal skin, mucosa and wounds with minimal irritation,sensitization or chemically induced discomfort at the effectiveconcentrations. Hydrogen peroxide defenses and enzymatic mechanisms ofplants are largely homologous and therefore compatible with mammiliancell defenses. Very low effective overall concentration dilutespotential sensitizing and irritating components to insignificant levels.The composition is also compatible with standards of wound care such ashydrogen peroxide, benzalkonium chloride, alcohol or saline used indebridement.

Further, some embodiments of the invention provide an antimicrobialcomposition that can maintain antimicrobial effectiveness at lowconcentrations in water and high levels of dilution into carriers suchas water, alcohol, propylene glycol, oil emulsions, fatty acidemulsions, hydrogels, or plant-derived bulking carriers, such as aloefor antiseptic formulations for injuries to human and animal skin, andmucosa to provide positive benefit during the initial stages of healing.A preferred method of production produces a high biocidal potency watersoluble composition without secondary concentrating processes.

Some embodiments of the invention provide a mycocidal composition and anenhanced mycocidal composition with the addition of non-oxidativemycocidal co-ingredients such as essential oils, particularly theessential oils of citron, cinnamon, usnic acid, eucalyptus, oregano,almond, or the formulation with surfactants such as sodium laurelsulfate, or other ionic or non-ionic surfactants. The aggressiveoxidative nature of hydrogen peroxide is highly effective againstyeasts, such as Candida-albicans. Mycocidal effectiveness is limited atthe low concentrations against larger eukaryotic cells and fungi withhigh level of external catalase, such as Aspergillus niger. Sub-lethalconcentration can maintain mycostasis of Aspergillus niger, butco-ingredients as mentioned provide improved killing.

Diluted extracts can be used on surfaces, skin, underclothing, shoes,oral mucosa, or other moist porous environments to control bacteriainduced odor. Diluted extract can be administered orally to controlgastroenteritis or other bacteria induced gastric disorders in humansand animals. The plant species tested have a substantial history ofingestion for folk remedies. Some embodiments of the invention produce acomposition that is effective at sanitizing and preserving potable waterwithout the adverse toxicity, taste, and odor associated withchlorination. The preferred composition of oxidizing molecule complex oraggregates provides a stable, persistent antimicrobial activity ateffective dilutions comparable to or lower than free chemical oxidizingdisinfectants, such as chlorine.

The use of endogenous or added H₂O₂ to crosslink free solubleproteinaceous compounds that have been extracted from plant tissue. H₂O₂plays a key role in the normal lignification of plant cell walls andpolymerization of free soluble proteins released when plant cells aredamaged or stressed. This is a defensive adaptation in plants thatpotentially seals a wound or thickens the cell walls for a mechanicalbarrier to limit loss of liquids and deter pathogen access. Theenzymatic reduction of hydrogen peroxide is known to initiate thecross-linking and polymerization of soluble proteins to form dimers,trimers, and higher protein polymers. It is suggested that catalase andperoxidase catalyzes the oxidation of hydrogen donors to form freeradicals quinones or other potential intermediates, which subsequentlyinteract with, cross link, and alter the soluble proteins (Stahman etal., Biochem 3. 2000 Jul. 1; 349(Pt 1): 309-321). The harvesting,storage, dessication, mechanical reduction, and extraction processes allcause the loss of endogenous H₂O₂. Addition of H₂O₂ to the extractedsoluble proteins can initiate cross-linking, particularly in thepresence of intact peroxidase. The use of fresh, hydrated orpartially-dessicated plant tissues can contain a significant level ofendogenous H₂O₂. Cold processing consisting of macerating tissue andsoaking in cool water or cold pressing, and these processes releaseprotein fragments from ruptured and autolyzed cells along with somecontent of endogenous H₂O₂ that can promote formation of cross-linkedproteinaceous aggregates or complexes without addition of H₂O₂. The useof endogenous H₂O₂ does not necessarily eliminate the need for exogenousH₂O₂ in composition to saturate the binding sites. The use of syntheticchemicals, except H₂O₂, to initiate cross-linking is typicallyundesirable for natural product applications.

The use of cell wall lysates on intact tissues increases the availablefree compounds available for subsequent aggregate formation. This can beused to take the place of mechanical pulverization,

heating of the solution, or freezing to separate and disrupt cells andextract soluble proteins and enzymes.

Some embodiments of the invention provide a composition that can beproduced using a very small amount of raw plant material to allowsustainable and economical manufacture. The present method of productionrequires less than one milligram of dessicated whole plant mass toproduce a kg of antimicrobial solution at final dilution, but thoseskilled in the art will understand that the composition can be producedand used at higher and lower concentrations. This also facilitates batchblending of unprocessed dry plant stock to reduce variability related toseason, age, cultivation, and other factors.

The plant materials preferably used in production of this invention arecharacterized by high capacity to produce antimicrobial ROS, astringentpolymers or the combination as innate microbial defenses. Physicaladaptations to environmental stresses are often a good indicator ofthese.

The outer coverings of seeds and fruits tend to exhibit the ability towithstand exceptional environmental stress in protecting germinatingseedlings. Such adaptations are good indicators of the highly developedoxidative stress and pathogen management systems of these plants. Aparticularly environmentally durable H₂O₂-acting oxalate oxidase enzymeis commonly known as germin, a manganese containing homohexamer withboth oxalate oxidase and superoxide dismutase activities. It isprevalent in seeds, buds, and sprouts to provide protection during thevulnerable germination phases. Cereal grains are noteworthy forcontaining a high concentration of germins in roots and seed membranesto protect the seeds and seedlings during germination. The discardedhulls of the seed germs are a potential source of tissue useful withthis invention. An example is the cereal grains of the Poaceae family.

Succulent tissues of Aloe, Pachycereus, and Opuntia were selected fortheir history in folk medicinal use. Their characteristics areconsistent with xeric plants with high ROS defensive chemistry content,and are available without environmental, regulatory or cultivationconcerns. These succulants have structures adapted to retain a largequantity of water and asexually reproduce through stem/stalk cuttings.Their tissues have rapid lignification ability and high endogenous H₂O₂storage capacity suggesting a high population of peroxisomes andmembrane bound ROS management enzymes.

Plant tissues known for high polyphenol content also are good candidatesfor the plant sources. Examples are the barks and leaves of the Fagoseaeand Theaceae families.

Antimicrobial compositions of this invention have been produced fromtissues of the plant families: Agavaceae, Cactaceae, Poaceae, Theaceae,Leguminosae, Fagoseae and Lythraceae, However, the present invention isnot limited to these families. A person skilled in the art willunderstand that the polymers, structures and enzymes capable of binding,incorporating, sequestering or reacting with ROS or ROS producingmaterials, and other proper material are able to be used as embodimentsof the present invention. Specific examples of common plant tissues thathave been successfully used by the inventors in the production of thiscomposition include: wheat husk, barley germ, rice hull, variouscolumnar cacti, pomegranate husk, green tea leaves, aloe vera leaves,mung beans and carrot. The preferred plants are commercially cultivatedspecies that are generally regarded as safe (GRAS) by the US FDA thushaving a history of low toxicity.

Regardless of the antimicrobial mechanism, the suitability and potencyof a plant materials is subject to many species dependent, seasonal andcultivation factors. A major advantage of the present invention is theconsistent level of potency that can be achieved by sub-saturation ofthe plant material binding sites. The addition of H₂O₂ or othersubstrate at a molar quantity below the minimum baseline that the plantmaterial can be assured to bind, provides dose control and qualitymetrics that are rare in natural products.

The method of complexing a large number of bioactive molecules canproduce enhanced antimicrobial effectiveness at lower concentrationsthan equivalent dilution of free active components. An antimicrobialcomposition is provided that can be an effective plant based biocide atbelow 10 ppm molar concentration of the bioactive raw ingredient. In thecase of hydrogen peroxide as the bioactive component, this results inlow native toxicity and minimum accumulation concern levels tofacilitate regulatory approvals as a preservative additive for foods,cosmetics, and medicines. Other concentrations may be used asappropriate for the applications. Bacterial inhibition in aqueoussolutions has been demonstrated at concentrations as low as 10 parts perbillion. The ability to remain biocidally functional at such lowconcentrations is valuable in uncontrolled dilution situations such asfor aquafarms, surface water or ground water remediation, agriculturalsanitation, drinking water decontamination and industrial waterprocessing.

Embodiments of the Present Invention

Embodiments of the present invention are directed to a method of formingand the composition of a plant-based biocidal solution. The plant-basedbiocidal solution contains a bioactive material and a plant-basedcomplex. In some embodiments, the bioactive material can be an oxidizingsubstrate capable of releasing reactive oxygen species. Reactive oxygenspecies (ROS) are ions or very small molecules that include oxygen ions,free radicals, and peroxides, both inorganic and organic. They arehighly reactive due to the presence of unpaired electrons. In someembodiments, the bioactive material is hydrogen peroxide. In someembodiments the bioactive material can be, but is not limited to, ozone,fatty acid peroxides, other peroxides, halogens, antibiotics, or otherbioactive compounds that can be performance enhanced by stabilizationand concentrated by the plant compound complex.

The plant-based complex can be formed from a cellular material of aplant cell, a plant cell fragment or fragments, a single or network ofmultivalent plant polymer or oligomer, or a combination of cellularfragments and plant polymers or oligomers capable of binding, fixing,sequestering, attracting or incorporating multiple instances ofbioactive molecules or radicals in a manner that maintains theirbio-reactive function. Binding, fixing or incorporating refers to anychemical bonding including covalent, ionic, Van der Waals, or hydrogenbonds, electrostatic attraction, enzymatic retention, entrapment,entanglement or other mechanism that immobilizes the bioactive componentto the plant material. This includes reversible and non-reversiblechemical reactions that incorporate the bioactive material, adegradation product a molecular subunit or ROS.

FIG. 2 illustrates a plant-based biocidal solution 200 of the presentinvention. The plant-based biocidal solution 200 contains a plant-basedsubstance 204. The plant-based substance 204 has cites 210 to bindbioactive material 202, including hydrogen peroxide 208. The plant-basedsubstance 204 can be formed from a cellular material 212 of a plant 206.In some embodiments, the plant-based biocidal solution 200 contains asolvent. The solvent can be water, organic solvent, inorganic solvent,gas, supercritical fluid, or any combination thereof.

In some embodiments, the plant-based complex 204 is formed from thecellular material 212 in the form of any of the following singularly orin combination: (1) cellular fragments with attached molecules withcompatible bioactive material binding sites 210, (2) partially or whollyintact plant cellular structures, membrane fragments, invagintions ororganelles populated with bioactive material binding capable molecules,(3) proteinaceous aggregates or fibrils of intact, partially denaturedor fragmented enzymes, or other proteins with the ability to bindbioactive material in a bio-reactive state, and (4) other solublecompounds that may provide useful independent activity, synergisticactivity or act as reaction co-factors. Alternatively, in someembodiments, plant cellular material 212 is peroxosomes, mitochondriaand cell membranes, which are sites of H₂O₂ generating and regulatingenzymes. In some embodiments, the sites 210 are bioactive materialrelated enzymes, such as catalases, peroxidases, oxalate oxidases,glucose oxidases, and dismutases, having the ability to capture and/orcatalyze reactions with bioactive material. Even if inactive in theiroriginal function, the structure of these molecules can provide lowenergy binding sites for fixing bioactive material while preserving itsbio-reactivity.

In some embodiments, the bioactive material 202 can be a substrate of amolecule or compound that generates reactive oxygen species whentriggered by a catalyst, enzyme or other co-factor. Alternatively, thebioactive material 202 can be hydrogen peroxide 208. Some hydrogenperoxide can be obtained from an endogenous source in the plantmaterial. The exogenous addition of hydrogen peroxide can be obtaineddirectly from commercially available sources. In some embodiments, theexogenous commercial hydrogen peroxide 208 has a concentration of1-100%. In some embodiments, the concentration of hydrogen peroxide is1-50%. In some embodiments, the hydrogen peroxide concentration is20-50%. A sufficient concentration is necessary to accommodate the watercontent of the aggregate solution to allow subsequent dilutions. Theconcentration must create a sufficiently high diffusion gradient aroundthe plant material to overcome any remaining H₂O₂ degrading enzymes.

The concentrated exogenous H₂O₂ added to the concentrated aqueoussuspension/solution of the plant material provides for an higherdiffusion gradient to substantially overcome any diffusion and chargerepulsive gradients in the plant material complex to achieve targetsaturation of the H₂O₂ binding sites in an economically efficientproduction time.

In some embodiments, the endogenous expression or production ofendogenous bioactive material 202, binding enzymes or desiredmultivalent plant molecules can be enhanced by mechanical, abiotic orbiotic stress to the metabolically active plant source or tissue.Pathogen attack, environmental stress and mechanical trauma to inducepre-harvest stress and post harvest cutting into metabolically activeplant structures stimulate defensive responses that may provide forincreased production of desired plant material such as polyphenols,peroxidases or lignin forming precursors.

Alternatively, in some embodiments, the bioactive material 202 can alsobe generated by the treatment of the plant material solution with ozone(O₃). Ozone can be used alone or in combination with exogenous H₂O₂.Direct aeration of the plant material solution with O₃ from a commercialsource or a electrical Ozone generator dissolves into the solution forincorporation. Dismutases in the composition enzymatically convert H₂Oand O₃ into H₂O₂ and O₂. This method can be more difficult to control,but it is desirable in circumstances where concentrated H₂O₂ is notavailable.

In another alternate embodiment, the plant material can be combined as akit with a solid form of a bioactive material, in particular a hydrogenperoxide generating material such as sodium perchlorate, urea peroxide,potassium perchlorate to form the bioactive complex. The introduction ofthe kit into water will cause the incorporation of the kit materialsinto the biocidal complex. This method has the advantage of being morecompact and stable than aqueous solutions.

In further embodiments, the methods of minimizing premature degradationof bioactive material 202 for production are disclosed. Usingtemperature or dessication of the plant materials inactivatesH₂O₂-degrading enzymes prior to exogenous H₂O₂ addition. Catalases andperoxidases are the primary H₂O₂-degrading catalysts that must besubstantially inactivated from the plant material prior to combinationwith H₂O₂. Blanching of fresh plant matter or freezing/thawing of thefully hydrated plant cells or extended of heating solution with 1% orgreater NaCl can also effectively inactivate enzymes.

Another aspect of the invention uses the above described inactivatedH₂O₂-acting enzymes of the plant material as a method of capturing andstabilizing exogenously added H₂O₂ in a bioreactive form. Despite agreat deal of research on their unique biochemical characteristics, verylittle is known about the catalytic mechanisms of oxalate oxidases,dismutases, peroxidases and catalases. Recently theories on catalyticmechanisms of H₂O₂ acting enzymes suggest three dimensional tunnel-likestructures with some electrostatic guidance to the active site of ametal ion of Cu, Mn, or Zn. Minor perturbations in the three dimensionalstructure or absence of an enabling co-factor or monomer can render theenzyme functionally inactive or hypoactive but still capable of bindingthe hydrogen peroxide in a manner useful for the purposes of thisinvention.

Aqueous solutions of these plant materials combined with H₂O₂ exhibitedincreased bactericidal effectiveness of equivalent concentrations ofaqueous plant extracts without exogenous H₂O₂ addition and extendedduration than H₂O₂ alone. Test microbes were wild strain EscherichiaColi, Escherichia Coli ATCC 4352, Staphylococcus aureus ATCC 6538,Psuedomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231, andAspergillis niger ATCC 16404.

The present invention composed of various plant bases with H₂O₂ werechallenged in-vitro with a range of microbial titrations in water. In 11of 12 cases wild strain E. coli and S. aureus exhibited clean plateresults down to single parts per billion concentrations with a shallowramp in the time to complete kill vs dilution. Complete lack ofsurvivors or rebound in the microbe population was observed untildilution reached a point of apparent depletion. The 150 μg/l compositionfrom columnar cactus maintained 100% killing rate at 2 days and 7 daysuntil 107 concentrations of E. coli were reached. At that inflection theslope of the concentration vs. survival curve looked similar toconventional free compounds, though still at several orders of magnitudelower concentration. The extended flat line of the clean plate dosagewith the sudden steep transition from complete kill to survival isconsistent with first order kinetics consistent with a “concentratedpacket” behavior between the bacteria and the biocidal composition.

The composition proved only mycostatic on Aspergillus niger. Thelocalized oxidative capacity of the reactive complexes tested wasinsufficient to overcome the considerable catalytic neutralizationcapacity of this exceptionally large eukaryote pathogen. However,absolute mycostasis at the bacteriostatic concentrations is consistentwith the ability to overcome the smaller defense capacity of theAspergillus buds. Bio-reactivity against virus, amoeba, and their cystsis consistent with the oxidative performance of hydrogen peroxide.

The plant materials 206 can be obtained from the tissues of most higherplants. The inventors have successfully produced the invention fromplants of the families of Agavaceae, Cactaceae, Poaceae, Theaceae,Leguminosae and Lythraceae. However, the present invention is notlimited to these families. Proper plants are able to be used inaccordance with present invention. In some embodiment, plants that aregenerally regarded as safe by the U.S. FDA having a history of lowtoxicity are used.

An example is the cereal grains of the Poaceae family. Many plants inthe family have a particularly high content of H₂O₂ acting compounds inthe normally discarded grain sheaths and roots. Various species of Aloe,Pachycereus, and Opuntia were selected for initial testing because oftheir history in human food or folk medicinal use, characteristicsconsistent with xeric plants with high H₂O₂ defensive chemistry content,and availability without environmental, regulatory or cultivationconcerns. Plants, such as these succulants, have structures adapted toretain a large quantity of water and asexually reproduce throughstem/stalk cuttings. Their tissues have an abundance of distributedlignification ability and related H₂O₂ acting structures, such asperoxisomes and membrane proteins, that function in both defensive andgrowth processes. Seeds and fruits also exhibit the ability to withstandexceptional environmental stress in protecting germinating seedlings.Such adaptations are good indicators of the highly developed oxidativestress and pathogen management systems of these plants. A particularlyenvironmentally durable H₂O₂-acting oxalate oxidase enzyme is commonlyknown as germin, a manganese containing homohexamer with both oxalateoxidase and superoxide dismutase activities. It is prevalent in seeds,buds, and sprouts to provide protections during the vulnerablegermination phases. Cereal grains are noteworthy for containing a highconcentration of germins in roots and seed membranes to protect theseeds and seedlings during germination. The discarded hulls of the seedgerms are a potential source of tissue useful with this invention.

Regardless of the binding mechanism, the suitability and potency of aparticular plant is affected by many species dependent, seasonal, andcultivation factors. One of the advantages of the present invention isthe consistent level of potency that can be achieved by sub-saturationof the plant material binding sites. The addition of H₂O₂ at a molarquantity below the minimum baseline that the plant material can beassured to bind provides dose control and quality metrics that are rarein natural products.

FIG. 3 illustrates a flow chart of a method of forming a plant-basedbiocidal solution of the present invention. The method of forming theplant-based biocidal solution of the present invention is achieved bycombining a plant-based complex 302 with a solution 304 containing abioactive material 306.

As described above, in some embodiments, the plant-based complex 302 andsolution 304 can be prepared by the same method as preparing theplant-based complex 204 and solution 200, respectively. The bioactivematerial is added or generated by the same method described above. Themethods of adding or generating the bioactive material, including butnot limited to: (1) exogenous adding hydrogen peroxide 308; (2) inducingexpression of increased H₂O₂ acting enzymes or structures throughmechanical abiotic stress 310; (3) degrading the added ozone (O₃) by anactive dismutase in the complex or solution, or in combination withdirect adding of the H₂O₂ 312; (4) using temperature or dessication toselectively inactivate H₂O₂ degrading enzymes prior to exogenous H₂O₂addition 314; (5) using temperature or dessication to inactivate allbioactive material acting enzymes prior to exogenous H₂O₂ addition 316;and (6) using inactivated H₂O₂ acting enzyme molecules to capture andstabilize free H₂O₂ in a bioreactive form 318.

FIGS. 4A and 4B illustrate a use of plant-based biocidal solution of thepresent invention. The plant-based biocidal solution 402, containing aplant-based complex 404 binding bioactive material 406, can be appliedto a target 408. In some embodiments, the plant-based biocidal solutioncan deliver high localized concentration of the bioactive material 406to the target 408. In some embodiments, the target 408 can be apathogen. In other embodiments, the plant-based biocidal solution 402can have beneficial effect to human wound 412 or animal wound 414healing.

FIG. 5 illustrates another use of plant-based biocidal solution in theform of microscopic clusters, complexes, and aggregates of the presentinvention. The plant-based biocidal material 502 forms a microscopiccluster 504, a complex 506, or an aggregate 508 from a suspension of theplant-based complex. The plant-based biocidal material 502 can beapplied to the target 510, thereby impairing the target 510.

Examples of Preparing the Composition

One example of the preparation of the composition is as following: largepieces of stalk or leaves are harvested from live plants, de-spined andwashed in cold water, and abiotic stress may optionally be induced bycutting the plant leaves or stalks into sufficiently large pieces topreserve sufficient local metabolic ability to express increaseddefensive enzyme structures. The pieces are then allowed to low-heat/airdessicate to inactivate the H₂O₂ degrading enzymes. Fine pulverizationof the dried plant pieces increases solubility and disruption of cellsto form fragments or subcellular particles. These cell fragments orparticles contain both water soluble and non-water soluble forms ofinactive materials, binding enzymes, potentially cooperating factors aswell as potentially undesirable compounds. Water is used as theextraction medium for its H₂O₂ compatibility and to minimize undesirablenon-water-soluble alkaloids, terpenes and the like that generally havehigher toxicity than water soluble compounds. The pulverized plantmaterials are mixed in room temperature or heated water in the ratio of100˜20,000 water to 1 plant and allowed approximately 24 hours fordissolving of soluble materials. The liquid is passed through a 5 micronfilter, then a concentration of 30-50% food grade H₂O₂ is added to theplant-water solution to make a final concentration of 0.05˜3% of H₂O₂,preferably 1%, and allowed to react for one hour to promotecross-linking of proteins into aggregates. Additional food grade H₂O₂with a concentration of 30-50% can be added to saturate availablebinding sites in the solution. The solution is allowed to react for aminimum 2 hours, preferably 8-24 hours. The solution is then diluted toreduce the introduced H₂O₂ content to 0.02%, a level at which theunbound H₂O₂ will degrade spontaneously within a few days. Thecomposition is diluted to desired commercial concentration and packaged.

Standard methods are used to evaluate and indicate specific minimumbiocidal potency and H₂O₂ content. The solution is sampled and dilutedto a concentration of 100 parts per billion.

Method 1: challenged with a solution of 106/ml indicator bacteriaculture. The plates must show zero colonies after 12 hours.

Method 2: the H₂O₂ may be also tested using catalase treated filterpaper. The treated disk is submerged in standard diluted solution andthe disk must become buoyant in less than specified time.

Method 3: the use of commercially available hydrogen peroxide teststrips provide colorimetric indication of bioactive hydrogen peroxidecontent.

The term binding or its equivalents are used to illustrate theinteractions between or among molecules. The interactions includechemical bonds and physical forces. For example, covalent interactions,ionic interactions, Van der Waals interactions, electrostatic orhydrogen bonds, reversible and irreversible chemical reactions,oxidation and reduction reactions, or other proper forces and reactions.

The present invention has been described in terms of specificembodiments incorporating details to facilitate the understanding ofprinciples of construction and operation of the invention. Suchreference herein to specific embodiments and details thereof is notintended to limit the scope of the claims appended hereto. It will bereadily apparent to one skilled in the art that other variousmodifications may be made in the embodiment chosen for illustrationwithout departing from the spirit and scope of the invention as definedby the appended claims.

We claim:
 1. An ingestible composition having hydrogen peroxide with anincreased half-life, the composition comprising: a combination of aningestible, water-soluble polyphenol as an extract from a plant, whereinthe extract has been heated to substantially inactivate hydrogenperoxide degrading enzymes; and, hydrogen peroxide, wherein the hydrogenperoxide was added to the extract after the extract was heated tosubstantially inactivate hydrogen peroxide degrading enzymes; wherein,the ingestible, water-soluble polyphenol is bound to the hydrogenperoxide in the combination; and, the bound ingestible, water-solublepolyphenol increases the half-life of the hydrogen peroxide in theingestible composition, the stability of the hydrogen peroxide in thecombination being greater in an aqueous solution than the stability ofthe hydrogen peroxide alone in the aqueous solution.
 2. The compositionof claim 1, wherein the composition comprises 1% of hydrogen peroxide.3. The composition of claim 1, wherein the composition comprises 0.05%to 3% of hydrogen peroxide.
 4. The composition of claim 1, wherein thecomposition comprises 0.02% of hydrogen peroxide.
 5. The composition ofclaim 1, wherein the composition is provided in a solid form, the solidform being more compact and stable than an aqueous solution of theextract and the hydrogen peroxide.
 6. The composition of claim 1,wherein the combination of the polyphenol and hydrogen peroxide is abiocide.
 7. The composition of claim 1, wherein the combination of thepolyphenol and hydrogen peroxide is a mycocide.